Three-dimensional visualization of the route of protein import: the role of nuclear pore complex substructures

The three-dimensional localization of nucleoplasmin and wheat germ agglutinin (WGA) at the nuclear envelope of Xenopus oocytes is demonstrated by microinjecting protein coated gold colloids and examining their distribution using both stereo transmission electron microscopy and field emission in-lens scanning electron microscopy. Localization of many WGA gold particles and nucleoplasmin gold particles at the same nuclear pore complex (NPC) following coinjection is demonstrated. Binding of the WGA gold in the central region of the NPCs appears to form a barrier, preventing the import of nucleoplasmin gold, and includes central localization along radial "tracks" which correspond to the internal filaments connecting the cytoplasmic ring and the central region of the NPC. We suggest that these filaments may in some way be involved in opening and closing of the central channel of the NPC for transport. Transport of nucleoplasmin through the central region of the NPCs appears to be in "single file" regardless of the size of the colloidal gold, and distribution into the nucleoplasm appears to be through the basket rings with no association of the nucleoplasmin gold with the basket filaments being observed.